Method for stabilizing aqueous phosphoenolpyruvic acid solution

ABSTRACT

Disclosed is a method for stabilizing an aqueous solution of phosphoenolpyruvic acid and its salt, which comprises placing the solution under the condition of pH 10 to 13. 
     Disclosed is also a container for phosphoenolpyruvic acid, comprising an outer bag comprising a polyvinyl chloride type resin and an inner bag comprising an alkali resistant resin for storing an aqueous solution of phosphoenolpyruvic acid or its salt under the condition of pH 10 to 13 therein. 
     According to the present invention, a method for stabilizing phosphoenolpyruvic acid in an aqueous solution and a container for housing the stabilized phosphoenolpyruvic acid accompanied therewith can be obtained.

BACKGROUND OF THE INVENTION

This invention relates to a method for stabilizing an aqueousphosphoenolpyruvic acid solution and a container therefor.Phosphoenolpyruvic acid (hereinafter called "PEP") is a compoundrepresented by the following formula: ##STR1## and it is generallyexpected to be used in the form of sodium salt as the blood preservativeand the medicament for activation (Journal of Transfusion Society ofJapan, 32, 310 (1986); Japanese patent application No. 127320/1986).

However, PEP is unstable in an aqueous solution and susceptible tohydrolysis. Particularly, as the treatment temperature is higher, thistendency becomes marked during high pressure sterilization in anautoclave.

Therefore, it has been desired to develop a means for stabilizing PEP inan aqueous solution and a container for housing the stabilized PEPaccompanied therewith.

SUMMARY OF THE INVENTION

PEP is ordinarily used together with citric acid, sodium citrate, sodiumdihydrogen phosphate, maltose, adenine, etc. but it is unstable underneutral acidic conditions. As the result of investigations by thepresent inventors, it has been clarified that stable storage of PEP canbe accomplished by placing an aqueous solution of PEP or its salt underthe condition of pH 10 to 13.

Also, the present inventors have found that a container, comprising anouter bag comprising a polyvinyl chloride type resin and an inner bagcomprising an alkali resistant resin for storing an aqueous solution ofphosphoenolpyruvic acid or its salt under the condition of pH 10 to 13therein can specifically accomplish stable storage of PEP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the container for PEP of thepresent invention.

FIG. 2 is an illustration showing one example of the blood bag systemfor attachment of the container for PEP of the present inventionthereto.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The salt of PEP to be used in the present invention may include, forexample, sodium salt, potassium salt, lithium salt, etc.

According to the present invention, an aqueous solution of PEP or itssalt is placed under the condition of pH 10 to 13, preferably under thecondition of pH 10 to 12, and particularly preferably under thecondition of pH 11 to 12.

The concentration of the aqueous solution of PEP or its salt is notparticularly limited provided that it is not more than the saturatedconcentration. As the base to be used for adjusting the pH of the aboveaqueous solution, for example, sodium hydroxide, potassium hydroxide,lithium hydroxide, etc. may be employed.

The aqueous solution of PEP or its salt stabilized according to themethod of the present invention can be adjusted to an appropriate pHduring usage and used for various uses.

Next, the container for PEP is to be described. In the prior art, as theblood bag, those made of polyvinyl chloride type resins have been usedfor good workability by fusion, flexibility and good storability ofblood cells.

However, polyvinyl type resins conventionally used for blood bag will bedenatured when contacted with a solution of pH 10 or higher. As thecontainer for PEP which is able to embody the above PEP stabilizingmethod, a structure making a polyolefin having alkali resistanceconnected to a blood bag may be conceivable, but due to extremely poorfusion of a polyolefin with polyvinyl chloride, a bag of a polyolefincannot be connected to a blood bag made of polyvinyl chloride.

Therefore, as the means capable of embodying the above storing method, anovel container for PEP being easy in workability and capable of housingan alkaline solution is required.

In the present invention, as the polyvinyl chloride type resin, theremay be included polyvinyl chloride, polyvinylidene chloride andcopolymers comprising vinyl chloride or vinylidene chloride as the mainstarting material.

As the alkali resistant resin, there may be included, for example,polyolefins such as polyethylene, polypropylene, etc. and copolymerscomprising an olefin such as ethylene, propylene, etc. as the mainstarting material.

FIG. 1 shows schematically the medicament container of the presentinvention.

In the present invention, the inner bag 2 may be either not fixed to theouter bag 1 or fixed by an appropriate means. Also, in the figures, 3shows a parent bag, 4, 5 and 6 child bags, 7 a sampling needle, 8 analkaline solution and 9 a neutral or acidic solution, respectively.

The medicament container of the present invention is required to have astructure such that the content of the inner bag 2 may be releasedduring usage and the alkaline solution housed therein may be mixed withthe neutral or acidic solution housed in the outer bag 1. That is,during storage of the container, it is required that the solutions inthe inner bag 2 and the outer bag 1 should not contact each other.

As the means for providing such a structure, for example, there may beincluded manufacturing of an inner bag 2 with a thickness which is madeextremely thinner than that of the outer bag 1 so that the inner bag 2may be punctured by pressing of the container by means of a pointed headprovided innerside of the outer bag 1; manufacturing of an inner bag 2having an edge with a cutting so that it can be readily torn off fromoutside of the outer bag 1; manufacturing of an inner bag 2 having astructure such that a stopper is provided on the inner bag 2 and thestopper of the inner bag 2 can be readily plucked out by bending of theouter bag 1.

Referring now to FIG. 2, the medicament container of the presentinvention is described when used for a blood bag.

In the inner bag 2, an aqueous solution of PEP or its salt (hereinaftercalled "first solution") is housed with its pH being adjusted to 10 to13, preferably 10 to 12, particularly preferably 11 to 12 as mentionedabove. As the salt of PEP, for example, sodium salt, potassium salt,lithium salt, etc. may be included. As the base to be used for adjustingthe pH of the above aqueous solution to 10 to 13, for example, sodiumhydroxide, potassium hydroxide, lithium hydroxide, etc. may be included.The concentration of the aqueous solution of PEP or its salt is notparticularly limited, provided that it is not higher than the saturatedconcentration.

In the outer bag 1, an aqueous solution of the components other than PEPand its salt (hereinafter called "second solution") is housed. Suchcomponents may include, for example, disaccharides such as maltose,sucrose; monosaccharides such as galactose, mannitol; citric acid,sodium citrate, sodium dihydrogen phosphate and adenine, etc., but acompound other than the above compounds which is suitable for storage ofblood and will not denature the outer bag comprising polyvinyl chloridetype resin may be also used.

The first solution and the second solution should preferably be adjustedpreviously to the respective pH's and volumes so that the pH when bothsolutions are mixed may become 5.5 to 7.5.

After the container for PEP manufactured as described above is subjectedto high sterilization, the inner bag 2 is opened, the solution obtainedby mixing of the first solution and the second solution is led to aparent bag 3, wherein said solution is mixed with an erythrocyteconcentrate or erythrocytes obtained by removal of plasma, leucocytesand platelets from an erythrocyte concentrate and the parent bag 3housing the mixed suspension is stored at 4° C., whereby erythrocyteswhich have an excellent function can be stored stably for a long term.

The use of the container for PEP is not limited to blood bag, but can beused widely for housing an alkaline solution and a neutral or acidicsolution as separated from each other.

The present invention is described in more detail by referring toExamples, but these Examples do not limit the scope of the presentinvention at all.

EXAMPLE 1

An aqueous 2% PEP solution of which pH was adjusted with 1 N NaOH wasstored at room temperature for 60 days and its stability was examined.

In the solution with pH=7.4, PEP was gradually decomposed. Thedecomposition rate were 0.8% and 1.4% after 28 days and 60 days,respectively. In the solution with pH=12, PEP was found to be stablewithout any decomposition observed after 60 days.

EXAMPLE 2

For confirmation of thermal stability depending on the liquid propertyof PEP, the following experiments were conducted. An aqueous 2% PEPsolution was prepared, adjusted in pH with lN aqueous sodium hydroxideand autoclaved (121° C., 30 min.), followed by measurement ofdecomposition percentage. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        pH       7      8      9    10    10.5 11  11.5 12                            ______________________________________                                        Decompo- 79.7   28.9   24.0 17.9  13.2 7.3 4.6  1.0                           sition                                                                        percentage                                                                    ______________________________________                                    

As the liquid property is migrated to alkaline region, stability isincreased until substantially no decomposition of PEP was observed atpH=12.

EXAMPLE 3

A container housing the first solution and the second solutionrespectively in the inner bag made of polypropylene and outer bag madeof polyvinyl chloride was prepared.

    ______________________________________                                        First solution                                                                          PEP                    1.57 g                                                 (dissolved in distilled water for                                             injection, adjusted to pH = 12 with 1N                                        NaOH and then the total volume was                                            made up to 20 ml)                                                   Second solution                                                                         Maltose                9.2 g                                                  Sodium citrate         2.0 g                                                  Citric acid            1.4 g                                                  Sodium dihydrogen phosphate                                                                          0.14 g                                                 Adenine                0.01 g                                                 (dissolved in distilled water for                                             injection and the total volume made up                                        to 130 ml)                                                          ______________________________________                                    

The container was subjected to high pressure sterilization in anautoclave at 121° C. for 20 minutes. The PEP amount after thesterilization was the same as before. After the sterilization, the innerbag was punctured. The pH after mixing of the first solution and thesecond solution was about 6.5. Fresh human erythrocyte concentrate in aparent bag was centrifuged, and the above mixture was added to 150 ml ofthe human erythrocyte concentrate obtained by removal of the plasma andthe buffy coat layer (the erythrocyte had a hematocrit value of 95%) toprepare an erythrocyte suspension. At this time, said suspension had ahematocrit value of about 45%).

The erythrocyte suspension as prepared above was stored together withthe container at 4° C. for 6 weeks. The erythrocyte suspension wassampled every one week for measurement of the contents of2,3-diphosphoglyceric acid (hereinafter called "2,3-DPG") and adenosinetriphosphate (hereinafter called "ATP") in erythrocyte. Measurement wasperformed after incubation for one hour after sampling. Also, the extentof hemolysis was similarly examined. The erythrocyte concentrate by useof the currently practiced CPD solution was served as the control group.The results are shown in Table 2 and Table 3. Table 2 shows an amount of2,3-DPG and ATP as a function of time by week. Table 3 shows a degree ofhemolysis as a function of time by week.

                  TABLE 2                                                         ______________________________________                                               Week    0     1      2    3   4    5    6                              ______________________________________                                        Applied  2,3-DPG   45    35   23   14  12   8    6                            example                                                                       of this                                                                       invention                                                                              ATP       6.2   6.8  6.3  6.0 5.7  4.9  4.2                          (μM/gHb)                                                                   Control  2,3-DPG   13    12   6    2   --   --   --                           (μM/gHb)                                                                            ATP       4.0   3.8  3.5  3.1 --   --   --                           ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Week      0       1     2     3    4     5   6                                ______________________________________                                        Applied example                                                                         12      21    22    28   37    50  59                               of this invention                                                             (μM/gHb)                                                                   Control   14      30    65    105  --    --  --                               (μM/gHb)                                                                   ______________________________________                                    

According to the storage method of the present invention, PEP and itssalt can be stored stably.

The container for PEP of the present invention has easiness ofworkability and also can house an alkaline solution and a neutral oracidic solution as separated from each other.

We claim:
 1. A method for stabilizing an aqueous solution ofphosphoenolpyruvic acid or its sodium salt, which comprises admixingNaOH with said aqueous solution in an amount such that the pH of theresultant solution is from 10 to
 13. 2. The method according to claim 1,wherein said pH is 10 to
 12. 3. The method according to claim 1, whereinsaid pH is 11 to
 12. 4. The method according to claim 1, wherein anaqueous solution of NaOH is admixed with said aqueous solution ofphosphoenolpyruvic acid or its sodium salt.
 5. The method according toclaim 2, wherein an aqueous solution of NaOH is admixed with saidaqueous solution of phosphoenolpyruvic acid or its sodium salt.
 6. Themethod according to claim 3, wherein an aqueous solution of NaOH isadmixed with said aqueous solution of phosphoenolpyruvic acid or itssodium salt.
 7. The method according to claim 1, wherein an aqueoussolution of NaOH is admixed with said aqueous solution ofphosphoenolpyruvic acid.
 8. The method according to claim 2, wherein anaqueous solution of NaOH is admixed with said aqueous solution ofphosphoenolpyruvic acid.
 9. The method according to claim 3, wherein anaqueous solution of NaOH is admixed with said aqueous solution ofphosphoenolpyruvic acid.
 10. The method according to claim 1, wherein anaqueous solution of NaOH is admixed with said aqueous solution of asodium salt of phosphoenolpyruvic acid.
 11. The method according toclaim 2, wherein an aqueous solution of NaOH is admixed with saidaqueous solution of a sodium salt of phosphoenolpyruvic acid.
 12. Themethod according to claim 3, wherein an aqueous solution of NaOH isadmixed with said aqueous solution of a sodium salt ofphosphoenolpyruvic acid.